GroEL Chaperonin Assisted Protein Folding: studied by PETFCS and SmFRET
Many newly synthesized unfolded polypeptides require assistance by molecular chaperones in order to reach their active folded states. The GroEL/GroES chaperonin system is one of the most important bacterial chaperonin systems, which fulfills an essential function in assisting the folding of cytosolic proteins. GroEL is a homo-tetradecameric double-ring assembly of 60 kDa subunits with two opposing central cavities stacked back to back. Upon binding of ATP and the co chaperone GroES, GroEL transiently encapsulates newly synthesized or misfolded substrate proteins, which then can fold to the native state while being shielded from the crowded cytosolic environment. The mechanism of GroEL remains controversial whether GroEL acts as a passive cage, merely providing an aggregation preventing micro compartment, or whether GroEL actively accelerates the refolding of a subset of substrate proteins by modulation of the folding energy landscape.
We resorted to a variety of single molecule fluorescence techniques to address this controversy. A novel and sensitive dual color fluorescence cross-correlation spectroscopy assay shows the absence of transient aggregates at single molecular concentration. Using a single molecule FRET (SmFRET) based assay, we show that the acceleration of substrate protein folding by GroEL is preserved under single molecule conditions, where aggregation is excluded. We apply photoinduced electron transfer fluorescence quenching correlation spectroscopy (PETFCS) to demonstrate that the acceleration of substrate protein folding is achieved by encapsulation of a dynamic folding intermediate inside the GroEL cavity. Furthermore, we provide evidence that modulation of the folding energy landscape is a result not only of steric confinement during encapsulation, but also of the net negative charge of the GroEL cage wall. Taken together, these results represent a substantial advance in our understanding of the mechanism of the prokaryotic chaperonin system and our findings suggest that GroEL, by an active chaperonin mechanism, promotes substrate protein folding by entropic destabilization of folding intermediates.